JAC Evaluation of daptomycin susceptibility testing by Etest and the effect of different batches of media

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1 Journal of Antimicrobial Chemotherapy (2001) 48, JAC Evaluation of daptomycin susceptibility testing by Etest and the effect of different batches of media Peter C. Fuchs*, Arthur L. Barry and Steven D. Brown The Clinical Microbiology Institute, 9725 SW Commerce Circle, Suite A-1, Wilsonville, OR 97070, USA One hundred and ninety-five Gram-positive bacteria representing 17 species were tested for susceptibility to daptomycin by broth microdilution and Etest methods. The geometric mean daptomycin MIC was 0.46 mg/l by broth microdilution tests and 0.73 mg/l by Etest. The concentration of calcium in 12 different batches of agar varied from 4 to 36 mg/l. Daptomycin Etest MICs varied inversely with the calcium concentration. Etest daptomycin MICs for quality control strains were within proposed quality control range on media with >20 mg/l of calcium. Monitoring the calcium levels of agar media by testing appropriate quality control strains is important for daptomycin Etests. Introduction Daptomycin is a novel lipopeptide antibiotic that exhibits good in vitro antimicrobial activity against most Grampositive bacteria. 1 5 Its spectrum of activity includes methicillin-resistant staphylococci, penicillin-resistant pneumococci and vancomycin-resistant enterococci, all of which are becoming increasingly prevalent worldwide. Daptomycin is currently undergoing Phase III trials with oncedaily dosing and is showing encouraging preliminary results. 6 The in vitro activity of daptomycin has been shown to be significantly affected by the concentration of calcium in the test medium. 2,7,8 For broth dilution susceptibility testing of daptomycin it has been recommended to supplement the Mueller Hinton broth to a physiological concentration of 50 mg/l of calcium. 7 Batches of Mueller Hinton agar (MHA) currently available may vary considerably in calcium content and daptomycin disc diffusion tests on MHA with low calcium levels produce significantly smaller inhibitory zone diameters than tests on MHA with c. 25 mg/l of calcium. 7 The daptomycin disc diffusion interpretive criteria and quality control (QC) limits proposed recently were based on tests performed on MHA with c. 25 mg/l of calcium. 7 The present study was designed to: (i) test the performance of daptomycin Etest strips on MHA with c. 25 mg/l of calcium in comparison with broth microdilution tests as the standard, and (ii) evaluate the effect of calcium concentration in the medium on Etest results by performing tests on 12 different batches of agar media that were available for susceptibility tests at that time. Calcium and magnesium concentrations in all 12 batches were measured and found to be variable. Materials and methods Microorganisms The 195 Gram-positive bacterial isolates represented 17 species, which are listed in Table 1. From these a subset of 20 isolates representing eight species was selected for Etests on 12 different batches of media 10 organisms each on media with and without 5% sheep blood. Antibiotics Daptomycin standardized powder and daptomycin Etest strips (AB Biodisk, Solna, Sweden) were provided by Cubist Pharmaceuticals, Inc. (Cambridge, MA, USA). Media For broth microdilution tests, cation-adjusted Mueller Hinton broth (Difco Laboratories, Detroit, MI, USA) was further supplemented to contain 50 mg/l of calcium. For testing fastidious species such as streptococci, 3% lysed horse blood was also added. Etests were performed on a batch of MHA with 24 mg/l of calcium, which was supplemented with 5% defibrinated sheep blood when testing *Corresponding author. Tel: ; Fax: ; cmi@hevanet.com 2001 The British Society for Antimicrobial Chemotherapy 557

2 P. C. Fuchs et al. Table 1. Daptomycin MICs by broth microdilution and Etest for 195 organisms MIC (mg/l) Geometric mean Microorganism No. Test a range 50% 90% MIC % 1 dilution b Corynebacterium jeikeium 10 BMD Etest Enterococcus avium 5 BMD Etest Enterococcus faecalis 12 BMD Etest Enterococcus faecium 13 BMD Etest Enterococcus gallinarum 5 BMD Etest Listeria monocytogenes 23 BMD Etest S. aureus 22 BMD Etest Staphylococcus epidermidis 13 BMD Etest Staphylococcus haemolyticus 10 BMD Etest S. agalactiae 12 BMD Etest Streptococcus group C 5 BMD Etest Streptococcus group F 5 BMD Etest Streptococcus group G 5 BMD Etest Streptococcus milleri 5 BMD Etest S. pneumoniae 18 BMD (Pen MIC 1.0 mg/l) Etest S. pneumoniae 10 BMD (Pen MIC 2.0 mg/l) Etest Streptococcus pyogenes 12 BMD Etest Streptococcus viridans group 10 BMD Etest a BMD, broth microdilution. b Percentage within 1 two-fold dilution of BMD MICs. fastidious organisms. For the multiple batch study, 11 batches of agar media with blood and 11 batches of agar without blood were compared. These included three batches of MHA produced by Becton Dickinson (Cockeysville, MD, USA), two batches each of MHA produced by Difco Laboratories, Accumedia (now Neogen, Baltimore, MD, USA) and Remel (Lenexa, KS, USA), one batch of MHA produced by Oxoid (Basingstoke, UK) and Criterion (Santa Monica, CA, USA) and one batch of Iso- Sensitest agar produced by Oxoid. Susceptibility tests Broth microdilution tests were performed by the method outlined by the NCCLS. 9 The concentrations of daptomycin tested represented 0.5 two-fold dilution intervals 558

3 Daptomycin Etest media effects ranging from 16 to mg/l. For Etest procedures, the manufacturer s instructions were followed. Quality control The following QC strains were tested at the same time as the test strains: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC and Streptococcus pneumoniae ATCC Calcium and magnesium levels were assayed (Beckman/Coulter Syncron CX7 with Arsenazo III as dye indicator) for each batch of media at the Chemistry Laboratory (Peter Anderson, Director) of Providence St Vincent Medical Center (Portland, OR, USA). Results Daptomycin MICs generated by the broth microdilution method were compared with those obtained by the Etest method (Table 1). For the entire group of 195 isolates the geometric mean MIC for broth microdilution tests was 0.46 mg/l, compared with 0.73 mg/l for Etest MICs. There was, however, some variation in the differences between Etest and broth microdilution MICs among the different species tested (Table 1). For example, Etest MICs were some 10% higher than broth microdilution MICs for some enterococcal species, but were nearly three-fold higher for Streptococcus agalactiae. For individual isolates the Etest MICs varied from 1.5 two-fold concentrations below to three two-fold concentrations above the broth microdilution MIC. Despite the clear tendency toward higher daptomycin MICs by Etest, 84% of Etest results were within one two-fold dilution of the broth microdilution MIC. The concentrations of calcium and magnesium in the test media are listed in Table 2. Etest MICs correlated inversely (R = 0.76) with the calcium concentration of the agar medium. Calcium concentrations ranged from 4 to 36 mg/l and the corresponding geometric mean daptomycin Etest MICs ranged from 11.7 to 0.44 mg/l for the 10 nonfastidious strains and 3.63 to 0.28 mg/l for the 10 strains tested on blood-containing media. Because of the inverse correlation (R = 0.70) of calcium and magnesium concentrations in the different agars, a modest positive correlation (R = +0.60) between magnesium concentration and daptomycin MICs was not unexpected. In general, MHA (with or without added sheep blood) containing >20 mg/l of calcium yielded daptomycin Etest MICs within one two-fold concentration of the broth microdilution MICs. Etest MICs on media with 20 mg/l of calcium were more than double broth microdilution MICs. The batch of Iso- Sensitest agar (a medium more commonly used in Europe) that we evaluated was also deficient in calcium and gave Etest daptomycin MICs more than eight-fold higher than broth microdilution MICs. Daptomycin Etest MICs for the three QC strains were consistently within the control range proposed previously 7 only when tested on agar media containing >20 mg/l of calcium (Table 2). All Etest QC results during the 195 isolate study that were performed on batches 7A and 7B were within the proposed QC range. Discussion Increasing the calcium concentration in the test medium has been reported previously to enhance the in vitro activity of daptomycin with both broth dilution and disc diffusion tests. 2,7,8 In the original study that led to the proposed daptomycin MIC breakpoints, tests were performed in cation-supplemented Mueller Hinton broth (with 50 mg/l of calcium), which was standard at the time. 3 Subsequently, calcium supplements were reduced to mg/l. Other reports have also recommended the 50 mg/l concentration of calcium for broth microdilution tests of daptomycin. 2,7 Although the calcium concentration in Mueller Hinton broth can be standardized and/or controlled with relative ease, incorporation into agar is more difficult to adjust. The calcium and magnesium content of current batches of MHA vary considerably (Table 2) and the concentration of calcium has been shown to have a significant effect on daptomycin disc diffusion zone diameters. 7 Most currently available batches of MHA in the USA conform to the manufacturer s performance test limits as published by the NCCLS. 10 However, that performance test does not include daptomycin discs, since it is still an investigational drug. To further judge cation content of the 12 batches of agar, 10 g gentamicin discs were tested in duplicate against Pseudomonas aeruginosa ATCC For all but one batch of MHA the zone diameters were within the control range. Only batch 2 (total cation content 22 mg/l) and the only batch of IsoSensitest agar yielded results outside the control range. If and when daptomycin is approved, it will be important that media manufacturers resolve this problem as quickly as possible. In the current study we have demonstrated that the calcium effect is also very pronounced with daptomycin Etests. We are unaware of other instances in which media variations have had such a significant effect on Etest MICs. Until MHA becomes standardized with respect to calcium concentration, this will be a challenge for the Etest manufacturer as well as Etest users. Although the number of QC tests in this study is too small to be conclusive, they do indicate that the QC strains S. aureus ATCC and E. faecalis ATCC may be used to identify media with insufficient calcium. The daptomycin MIC QC ranges proposed for broth microdilution tests appear to work satisfactorily for Etests on MHA with >20 mg/l of calcium. Routine testing of these QC strains will be important for Etest users who test daptomycin susceptibility of clinical isolates. 559

4 P. C. Fuchs et al. Table 2. Comparison of daptomycin Etest results by cation content of the agar media Cation level (mg/l) Etest MIC (10 strains) QC strain Etest MICs a CMI batch no. b Ca 2 Mg 2 geometric mean MIC difference from BMD c S. aureus E. faecalis S. pneumoniae A dil 0.5, ,1.5 11A dil 0.75, ,2.0 4A dil 0.5, ,4.0 7A dil 0.75, ,2.0 1A dil 1.5, ,3.0 3A dil 1.5, ,3.0 8A d dil 0.75, ,4.0 2A dil 2.0,1.0 12,6.0 9A dil 3.0,4.0 32,16 10A dil 3.0,6.0 48,32 6A e dil 2.0, ,192 Cation level (mg/l) Etest/BMHA (10 strains) QC strain Etest MICs a CMI batch no. b Ca 2 Mg 2 geometric mean MIC difference from BMD c S. aureus E. faecalis S. pneumoniae B dil B dil B d dil B dil B dil B dil B dil B dil B dil B dil B e dil a MIC values in mg/l; values out of control values previously established 7 are in bold; duplicate tests were recorded on agar without blood. b Batches labelled A and B for each CMI batch no. differed only by the presence of sheep blood in B. c Geometric mean broth microdilution MICs were 0.51 mg/l for the 10 non-fastidious strains and 0.17 mg/l for the 10 fastidious strains. d CMI batches 8A and 8B represent two different batches from the same manufacturer. e Batch 6 is the single batch of IsoSensitest agar tested, all others are MHA. 560

5 Daptomycin Etest media effects Acknowledgements This study was subsidized by a financial grant from Cubist Pharmaceuticals, Inc. References 1. Appelbaum, P. C., Spangler, S. K., Crotty, E. & Jacobs, M. R. (1989). Susceptibility of penicillin-susceptible and -resistant strains of Streptococcus pneumoniae to new antimicrobial agents, including daptomycin, teicoplanin, cefpodoxime and quinolones. Journal of Antimicrobial Chemotherapy 23, Eliopoulos, G. M., Willey, S., Reiszner, E., Spitzer, P. G., Caputo, G. & Moellering, R. C., Jr (1986). In vitro and in vivo activity of LY146032, a new cyclic lipopeptide antibiotic. Antimicrobial Agents and Chemotherapy 32, Jones, R. N. & Barry, A. L. (1987). Antimicrobial activity and spectrum of LY146032, a lipopeptide antibiotic, including susceptibility test recommendations. Antimicrobial Agents and Chemotherapy 31, Low, D. E., McGeer, A. & Poon, R. (1989). Activities of daptomycin and teicoplanin against Staphylococcus haemolyticus and Staphylococcus epidermidis, including evaluation of susceptibility test recommendations. Antimicrobial Agents and Chemotherapy 33, Swenson, J. M., Facklam, R. R. & Thornsberry, C. (1990). Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus, and Lactobacillus species. Antimicrobial Agents and Chemotherapy 34, Tally, F. P., Zeckel, M., Wasilewski, M. M., Carini, C., Berman, C. L., Drusano, G. L. et al. (1999). Daptomycin: a novel agent for Gram-positive infections. Expert Opinion in Investigational Drugs 8, Fuchs, P. C., Barry, A. L. & Brown, S. D. (2000). Daptomycin susceptibility tests: interpretive criteria, quality control, and effect of calcium on in vitro tests. Diagnostic Microbiology and Infectious Disease 38, Jones, R. N. (1989). Effects of reduced cation supplement recommendations (National Committee for Clinical Laboratory Standards) on daptomycin antistaphylococcal activity. Journal of Antimicrobial Chemotherapy 33, National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A5. NCCLS, Wayne, PA. 10. National Committee for Clinical Laboratory Standards. (1996). Protocols for Evaluating Dehydrated Mueller Hinton Agar: Approved Standard M6-A. NCCLS, Wayne, PA. Received 1 February 2001; returned 21 May 2001; revised 29 June 2001; accepted 19 July

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